Our genomes experience a large quantity of DNA damage, from both exogenous sources and endogenous byproducts of normal cellular processes. The ability of the cell to recognize and repair DNA damage is essential for maintenance of genomic integrity. Failures in DNA repair can lead to mutations, cell death, premature aging, and cancer. The work proposed here aims to analyze repair of a specific and highly deleterious type of DNA damage, a double-strand break (DSB), and to determine the mechanism by which DSBs are repaired in the context of a genetically tractable organism. DSBs are repaired by either non- homologous end joining, where the ends are modified and joined, or homologous recombination, where identical sequences (such as a homolog or sister chromatid) are used to as a donor template to repair the DSB and restore information lost at the break. Normal cells are highly sensitive to homology of the donor template, as recombination repair between diverged sequences is highly suppressed to avoid genome rearrangements and instability. However, the genetic components responsible for this suppression are not clearly delineated. Lastly, RecQ helicases are a family of proteins that have unique and overlapping roles in unwinding DNA substrates and maintaining genome integrity (including suppressing recombination between diverged sequences). The potential for redundancy of these family members within and across species has not been addressed in the context of DSB repair. Using molecular and genetic analyses in Drosophila melanogaster, two novel DSB repair assays will be utilized to 1) determine the mechanisms by which simple DSBs are repaired in the context of a whole organism, 2) delineate how recombination between diverged sequences is suppressed, and 3) establish functional redundancy of RecQ helicases within and across species. Importantly, to maintain goals of the AREA award mechanism, this project will give undergraduate researchers hands-on experience in a wide variety of molecular and genetic techniques and hypothesis-driven training, which will provide a valuable skill set for a future career in biomedical research and/or health-related careers.